U.S. Pat. Nos. 4,514,495 and 4,517,292 disclose a method for depositing (1) a solution of a growth-affecting substance and (2) a microbe-containing solution, in a programmed manner onto the surface of a culture medium so as to allow for their interaction. In typical applications, the growth-affecting substance would be deposited to produce a gradient of potencies as a function of radial distance from the center while the microbe-containing solution would be deposited to provide the same concentration at all the selected locations on the culture plate. This permits the determination of the effect of different strengths (potencies) of the growth-affecting substance on the behavior of the microbial population. Measures of behavior could be the presence or absence of visible--or instrumentally detectable--colonies, or the number and/or size of such colonies.
A test in which an interaction property is the change from presence to absence of signs of growth on the culture medium is referred to as an endpoint test, where the "endpoint" is defined by the potency at which the transition from growth to no-growth occurs. Application of the procedures disclosed in the above-referenced patents to endpoint tests is described in the Spiral System Instruments, Inc. of Bethesda, Md. Preliminary User Guide, titled "Determination of Antimicrobial Susceptibility by the Spiral Gradient Endpoint (SGE) Test" and dated June, 1985. In this test the growth-affecting substance is an antimicrobial agent, i.e., a substance causing growth inhibition, deposited by means of the Spiral Plater.TM., which is an instrument marketed by Spiral System Instruments, Inc. for depositing programmed gradients of solutions, to produce a radial gradient of potencies. The solution containing the test organism is deposited as a radial line on the surface by swabbing or by means of a mechanical inoculator. After incubation, there will be growth along the line of test sample deposited from the outside of the plate toward the center, stopping at the point along the line where the potency of the antimicrobial agent is sufficient to prevent visible growth. To quantify this effect it is necessary to determine the potency of the antimicrobial agent at the point of change from growth to no-growth.
A test in which the measure of the effect is the number of colonies developed as a function of the potency of the growth-affecting substance is the bacterial mutation assay, such as the popular Ames assay. In this test, the growth-affecting substance is the test compound, which is evaluated for its ability to produce mutations in selected bacterial strain(s). If a mutation occurs, then the cell will replicate and produce a colony (provided the compound is not toxic). The number of colonies is a measure of the degree of mutagenicity, which will vary with the potency of the compound to which the test strain is subjected.
The above-referenced patents are applied to bacterial mutagenicity testing by depositing a solution of the compound with the Spiral Plater.TM. to produce a radial gradient of potencies, and also depositing a solution containing the test strain with the Spiral Plater.TM., but at a uniform concentration along the spiral deposition track. A count is made of the number of colonies developed in discrete segments of the spiral track, e.g., for each complete spiral. To obtain the desired dose-response information, it is also necessary to determine the average potency of the growth-affecting substance in each of the segments for which a colony count is made. See "Development and Validation of an Automated Approach to Bacterial Mutagenicity Testing," V. Houk, S. Schalkowsky & L. Clayton, poster paper presented at the annual meeting of Environmental Mutagen Society, Mar. 27-31, 1988 at Charleston, S.C.
There is a fundamental difference between the manner in which a growth-affecting substance and microbes combine with the culture medium after their deposition. Thus, while the microbes remain at or near the surface independent of elapsed time, the growth-affecting substance will diffuse radially as well as vertically downward after its deposition on the surface of the culture medium. The amount (weight) present at the surface as a function of elapsed time is a complex function of the physical properties of the substance, the potency gradient, properties of the culture medium into which diffusion takes place and environmental parameters, e.g., temperature. The method described in the above-referenced patents does not consider the above factors, and does not attempt to quantitate the potency at the surface where the interaction takes place. Instead it assumed that the surface potency can adequately be represented by a computation of the average weight of growth-affecting substance per unit volume of culture medium in the entire column of culture medium below the point of interaction on the plate, derived from the known volume of solution deposited by the Spiral Plater.TM. at any location along the track, the known weight of the growth-affecting substance per unit volume in this solution, and the height of culture medium in the plate.
The above average potency only approximates the actual surface potency and is useful in implementing the method of the above-referenced patents mostly because of the insensitivity of the test methods which are being replaced by it. Thus, standard endpoint tests as well as bacterial mutagenicity tests utilize a series of two-fold dilutions of the growth-affecting substance to test its effect on the same concentration of microbes. There is thus a 100% difference between the potencies of adjacent measurements and, when adding measurement uncertainties, a sensitivity of .+-.100% is usually associated with such tests. There is thus room within this range of variation of .+-.100% to accommodate some variation due to diffusion in the average potency value referred to in the above-referenced patents. The benefits of time and materials reductions, derivable from the methods disclosed in the above-referenced patents, are thus available. However, to also improve upon the accuracy and utility of test methods used to quantitatively assess the effect of microbial interactions with growth-affecting substances, it is necessary to know with accuracy the potency of the growth-affecting substance at specific locations on the surface of the culture medium. This requires the inclusion of diffusion effects in the determination of potency.